Irs modulators

ABSTRACT

This invention is directed to a general method for the chronic treatment, potential cure, or prevention of various metabolic and related diseases in people, including diabetes, by modulating IRS2 activity in cells and tissues in the body. IRS1 and IRS2 are part of the insulin or insulin-like growth factor signaling pathway. By upregulating the levels or functional activity of IRS2, insulin is used more efficiently by the body to control nutrient levels. By upregulating IRS2 levels or functional activity in pancreatic β-cells, glucose sensing and insulin secretion are enhanced.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. provisional application60/437,377, incorporated by reference herein.

FIELD OF THE INVENTION

This is a general method for the prevention, induction of long termremission, or even the cure of various metabolic diseases and disordersin human beings and animals, including type 2 diabetes, by regulatingthe IRS2/IRS1 levels and signaling function in cells and tissues in thebody. IRS1 and IRS2 are part of the insulin or insulin like growthfactor signaling pathways, but also mediate some signals through othergrowth factors and cytokines, including IFNγ, IL2, IL4, IL7, IL9, IL13or IL15, growth hormone, prolactin, or leptin. IRS1 or IRS2 functionalactivity also integrate signals emanating from proinflammatorycytokines, including TNFα, IL6, IL1β and related factors. In generalproinflammatory cytokines inhibit IRS1/IRS2 signaling which mightcontribute to insulin resistance syndromes.

BACKGROUND OF THE INVENTION

Diabetes mellitus is a complex and life threatening disease that hasbeen known for more than 2000 years. It occurs in mammals as diverse asmonkeys, dogs, rats, mice and human beings. The discovery of insulin andits purification in 1921 for use in people provided a partial treatmentfor diabetes that is still in widespread use today. Insulin levels areordinarily adjusted by the body on a moment to moment basis to keep theblood sugar level within a narrow physiological range. Periodic insulininjections, however, can only approximate the normal state because thecellular response to insulin in many cases is also reduced.Consequently, for these and other reasons which will be discussed indetail below, life threatening complications still occur during thelifetime of treated diabetic patients, especially in the case of type 2(adult-onset) diabetes.

Diabetes arises from various causes, including dysregulated glucosesensing or insulin secretion (Maturity onset diabetes of youth; MODY),autoimmune-mediated β-cell destruction (type 1), or insufficientcompensation for peripheral insulin resistance (type 2). (Zimmet, P. etal., Nature 414:782-787 (2001)). Type 2 diabetes is the most prevalentform of the disease: It is closely associated with obesity, usuallyoccurs at middle age, and afflicts 18.2 million Americans. The commonmechanism causing peripheral insulin resistance and β-cell failure isimportant to understand.

Peripheral insulin resistance contributes to type 2 diabetes, but β-cellfailure is an essential feature of all types of diabetes. β-cellsfrequently fail to compensate for insulin resistance, apparently becausethe IRS2-branch of the insulin and IGF signaling cascade which mediatesinsulin signaling in target tissues also is essential for β-cell growth,function and survival. (Withers, D. J. et al., Nature 391:900-904(1998)).

Because insulin resistance is a cause of metabolic dysregulation anddiabetes, understanding its molecular basis is an important goal.Genetic mutations are obvious sources of life-long insulin resistance,but they are associated with rare metabolic disorders and thus difficultto identify in the general population. Inflammation is associated withinsulin resistance and provides a framework to understand how diet,acute or chronic stress, and obesity might cause insulin resistance.Proinflammatory cytokines, including IL6 and tumor necrosis factor-α(TNFα) that are secreted from leukocytes during inflammation, are alsoproduced in adipose tissue. TNFα promotes serine phosphorylation ofIRS-proteins, which correlates closely with insulin resistance.(Hotamisligil, G. S. et al., Science 259:87-91 (1999); Hotamisligil, G.S. et al., Science 271:665-668 (1996); Peraldi, P. et al., J. Biol.Chem. 271:13018-13022 (1996)). Although TNFα regulates various kinases,the NH₂-terminal Jun kinase (Jnk) is a prominent effector because itbinds to IRS1 and IRS2 and phosphorylates serine residues that inhibitthe interaction between IRS1 and the insulin receptor. (Aguirre, V. etal., J. Biol. Chem. 277:1531-1537 (2002)). The knockout of Jnk1 in obesemice, or general inhibition of serine kinases by high doses ofsalicylates reduces Ser phosphorylation of IRS1 and reverseshyperglycemia, hyperinsulinemia, dyslipidemia in obese rodents bysensitizing insulin signaling pathways. (Fruebis, J. et al., Proc. Natl.Acad. Sci. U.S.A 98:2005-2010 (2001); Yuan, M. et al., Science293:1673-1677 (2001)).

Ubiquitin-mediated degradation of IRS-proteins also promotes insulinresistance (FIG. 1). IL6 secreted from leukocytes and adipocytesincreases expression of SOCS1 and SOCS3, known for the ability tosuppress cytokine signaling. Another function of SOCS1 and SOCS3 is torecruit an elongin BC-based ubiquitin ligase into the IRS-proteincomplex to mediate ubiquitinylation. Thus, ubiquitin-mediateddegradation of IRS-proteins might be a general mechanism ofcytokine-induced insulin resistance that contributes to diabetes orβ-cell failure. (Krebs, D. L. et al., Sci.STKE. 2003, E6 (2003)). Moderngenomic approaches have revealed new cytokines secreted directly fromadipocytes that directly influence nutrient homeostasis and insulinsensitivity, including leptin, adiponectin, resistin and others thatwill reveal new mechanisms to modulate insulin sensitivity.

The activity of protein or lipid phosphatases, including PTP1B, SHIP2 orpTEN modulates insulin sensitivity (FIG. 1). Disruption of each of thesegenes in mice increases insulin sensitivity, suggesting that each mightbe a target for inhibitor design. PTP1B resides in the endoplasmicreticulum where it dephosphorylates the insulin receptor duringinternalization and recycling to the plasma membrane. (Haj, F. G. etal., Science 295:1708-1711 (2002)). This specialized mechanism appearsto limit unwanted side effects associated with inhibition ofphosphatases, including unregulated cell growth.

The insulin receptor is the prototype for a family of homologousintegral membrane proteins composed of an extracellular insulin-bindingdomain that controls the activity of an intracellular tyrosine kinase. A150-kb gene on chromosome 19 composed of 22 exons encodes the humanproreceptor. During translation, two homologous pro-receptors form adisulfide-linked dimer that is glycosylated and cleaved to form aheterotetramer of two extracellular α-subunits and two trans-membraneβ-subunits. Insulin binds to the juxtaposed α-subunits facilitating ATPbinding and tyrosine autophosphorylation of the β-subunit, whichactivates the kinase and recruits cellular substrates to initiate signaltransduction. The full insulin-signaling pathway as presently known issummarized in the STKE Connections Map. (White, M., Insulin SignalingPathway, Sci. STKE Connections Map, as seen November 2003,http://stke.sciencemag.org/cgi/cm/CMP_12069).

Selective insulin binding is complicated by tissue-specific alternativesplicing that directs synthesis of two insulin receptor isoforms (IRaand IRb), and by post-translational assembly of hybrids between theseisoforms and the homologous IGF1 receptor (IGF1R). (Frasca, F. et al.,Mol. Cell Biol. 19:3278-3288 (1999)). IRb exclusively binds insulin,whereas IRa binds both insulin and IGF2 with similar affinities:Dysregulated splicing alters fetal growth patterns and contributes torare forms of insulin resistance in adults. (Frasca, 1989; Savkur, R. S.et al., Nat. Genet. 29:40-47 (2001)) Moreover, hybrid receptors composedof an αβ-dimer from the IGF1R and the IRb selectively bind IGF1, whereashybrid receptors composed of IGF1R and IRa bind IGFs and insulin withsimilar affinities (FIG. 1).

The first member of the insulin receptor substrate family of proteinswas discovered in 1985, and subsequent research efforts revealed theexistence of related IRS family members as well as the signalingpathways to which the IRS proteins are linked. After the discovery thatthe Insulin Receptor (IR) possessed a tyrosine kinase enzyme activity,many groups searched for insulin receptor substrates that might regulatedownstream signaling from the receptor. The first evidence for theexistence of an actual target protein for the Insulin Receptor,subsequently named an Insulin Receptor Substrate, or “IRS” protein,resulted from the use of phosphotyrosine antibody immunoprecipitateswhich surprisingly revealed a 185-kDa phosphoprotein (pp185) ininsulin-stimulated hepatoma cells. (White, M. F. et al., Nature318:183-186 (1985)). Purification and molecular cloning of pp185revealed one of the first signaling scaffolds as well as the firstInsulin Receptor Substrate protein (IRS1). (U.S. Pat. No. 5,260,200;Sun, X. J. et al., Nature 352:73-77 (1991)). IRS1 was determined to bebiologically important because it was phosphorylated immediately afterinsulin stimulation, and catalytically active insulin receptor mutantsthat failed to phosphorylate IRS1 were biologically inactive. Most, ifnot all, insulin signals are produced or modulated through tyrosinephosphorylation of IRS1, IRS2 or its homologs; or other scaffoldproteins including SHC, CBL, APS and SH2B, GAB1, GAB2, DOCK1, and DOCK2.Although the role of each of these substrates merits attention, workwith transgenic mice suggests that many insulin responses, especiallythose that are associated with somatic growth and carbohydratemetabolism, are mediated through IRS1 or IRS2.

IRS-proteins are composed of multiple interaction domains andphosphorylation motifs, but appear to lack intrinsic catalyticactivities. All IRS-proteins contain an NH₂-terminal pleckstrin homology(PH) domain adjacent to a phosphotyrosine-binding (PTB) domain, followedby a COOH-terminal tail with numerous tyrosine and serinephosphorylation sites. The PTB domain binds directly to thephosphorylated NPXY-motif—Asn-Pro-Xaa-Tyr(Pi), Xaa represents any aminoacid—in the activated receptors for insulin, IGFs or interleukin-4(IL4); the PH domain also couples IRS-proteins to activated receptors,but the mechanism is unclear. (Yenush, L. et al., Mol. Cell Biol.18:6784-6794 (1998)). Other receptors also recruit and phosphorylateIRS-proteins, including those for growth hormone, IL-9, IL-13 and IL-15,and various integrins. (Shaw, L. M., Mol. Cell Biol. 21:5082-5093(2001)).

Tyrosine phosphorylation sites in IRS1 and IRS2 bind common effectorproteins, including enzymes (phosphoinositide 3-kinase, the phosphataseSHP2, or the tyrosine kinase fyn) or adapters (SOCS1, SOCS3, GRB2, NCK,CRK, SHB and others).

Activation of PI3K during association with IRS proteins increases theactivity of protein kinase B (PKB), which phosphorylates varioussubstrates including BAD (important for cell survival), GSK3β(regulating growth and glycogen synthesis), and Foxo1 (controlling geneexpression) (FIG. 1). A role for Foxo1 in insulin or IGF action wasrevealed by mutations in the C. elegans ortholog Daf16. (Ogg, S. et al.,Nature 389:994-999 (1997)). During insulin or IGF stimulation, Daf16 andFoxo1 are phosphorylated by PKB and accumulate in the cytosol. Nuclearexclusion of Foxo1 inhibits hepatic gluconeogenesis, but stimulatesadipocyte differentiation and pancreatic β-cell function. (Nakae, J. etal., Dev. Cell 4:119-129 (2003); Kitamura, T. et al., J. Clin. Invest110:1839-1847 (2002); Puigserver, P. et al., Nature 423:550-555 (2003)).

IRS1 contains many tyrosine phosphorylation sites that arephosphorylated during insulin and insulin-like growth factor 1 (IGF1)stimulation, and bind to the Src homology-2 domains in various signalingproteins. The interaction between IRS1 and p85 activates the class lAphosphotidylinositide 3-kinase, thereby revealing the first insulinsignaling cascade that could be reconstituted successfully in cells andtest tubes.

Several experiments suggested that other related proteins might exist:IRS1 antibodies did not react completely with the phosphotyrosinecontaining protein that migrated at 185 kDa during SDS-PAGE; FDCP1 cellscontained a protein with characteristics similar to those of IRS1 butfailed to react with antibodies directed against IRS1; the liver oftransgenic mice lacking IRS1 still contained a protein in liver that hadcharacteristics of IRS1. All of these finding led to the purificationand cloning of Insulin Receptor Substrate 2 (IRS2), a second member ofthe IRS family. (U.S. Pat. No. 5,858,701; Sun, X. J. et al., Nature377:173-177 (1995)).

Experiments in transgenic mice revealed involvement of IRS1 and IRS2 inpromoting somatic growth and nutrient homeostasis. Without IRS1, miceare 50% smaller than normal from birth until they die at 2 years of age.Mice without IRS1 have less body fat and are glucose intolerant. Inmice, IRS2 is important for peripheral insulin action, as mice lackingIRS2 display glucose intolerance and hyperlipidemia.

Disruption of the IRS2 gene in mice using standard gene knockoutapproaches results in diabetes that develops during the first 10 to 12weeks of age. Pancreatic β-cells are lost from these mice as they age,and genes that are important for β-cell function are down regulated inmice lacking IRS2.

SUMMARY OF THE INVENTION

It has now been discovered that a family of target proteins thatfunction immediately downstream of the insulin receptor or insulin likegrowth factor receptors, termed the insulin receptor substrate (IRS)protein family, is of central importance in mediating the effects ofinsulin on responsive cells. In particular, up regulation of the levelor functional activity of IRS2 in humans will result in atherapeutically effective chronic treatment for patients suffering fromdiabetes, especially the adult onset (type 2) form of the disease, aswell as for other disorders in which IRS protein function isinsufficient, abnormal or absent altogether. Further, IRS1 and IRS2 areof central importance in the insulin or insulin like growth factorsignaling pathway, and also mediate signals by other growth factors andcytokines.

Accordingly, the invention is directed to a general method for thetreatment, cure, or prevention of various metabolic and relateddisorders, including diabetes, by regulating the level or functionalactivity of IRS proteins.

In one embodiment, the invention is directed to restoring or enhancinginsulin sensitivity in a cell by upregulating IRS2 function. Theinvention is further directed to enhancing pancreatic β-cell function byupregulating IRS2 function. According to the invention, a disease ordisorder characterized by reduced or insufficient signaling through IRS2can be treated by upregulating IRS2 function. Such diseases include, butare not limited to metabolic disease, diabetes, dyslipidemia, obesity,female infertility, central nervous system disorders, Alzheimer'sdisease, and disorders of angiogenesis.

According to the invention, upregulation of IRS2 function includesactivation of IRS2 or a complex that includes IRS2. Upregulation of IRS2function is also accomplished by inhibition of phosphorylation ofcarboxy terminal serine residues of IRS2. Upregulation of IRS2 functioncan be accomplished by enhanced expression of IRS2 or by inhibition ofdegradation of IRS2.

In another embodiment, the invention is directed to a method ofdetermining whether a small molecule is an activator or an inhibitor ofIRS2. In a cell-based assay, a Test Cell is provided which overproducesIRS2 and exhibits an increase in binding of an IRS2-binding protein toIRS2, relative to a Control cell which produces IRS2 at a lower level,or does not produce the protein at all, and which exhibits a lesseramount of binding of said protein to IRS2. Small molecules that activateor inhibit IRS2 are identified by measuring the amount of the IRS2binding protein bound to IRS2.

In another embodiment, the invention is directed to a method ofidentifying a small molecule capable of increasing the level ofexpression from an IRS2 promoter in a mammalian cell. In one suchembodiment, a Test Cell is constructed which contains a constructcomprising an IRS2 promoter operably linked to a reporter gene such thatincreased expression of the IRS2 promoter sequence using a substanceknown to be capable of upregulating the endogenous IRS2 gene results inan increase in said measurable characteristic of the Test cell. Smallmolecules that increase IRS2 expression are identified by detecting anincrease in reporter gene activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts components of the IRS signaling cascade.

FIG. 2 depicts aspects of the IRS2 signaling cascade that are specificto β-cells.

DETAILED DESCRIPTION

This invention pertains to generalized methods of preventing, curing orinducing durable long-term remissions in patients with diabetes,metabolic disorders, central nervous system diseases, obesity, fertilityand other human disorders in which an inappropriate level of functionalcellular activity of the IRS family of proteins contribute to thedisease state. The invention is particularly concerned with IRS2 andmodulation of the activity of IRS2-mediated cellular signaling pathwaysas a mechanism for treating human disease.

The invention is based on the recognition that the IRS2 branch of theinsulin/IGF signaling system coordinates important biochemical reactionsand signaling pathways needed for proper function of peripheral insulinsensitive tissues and cells (liver muscle and fat), the function ofcomponents in the brain that coordinate nutrient homeostasis andappetite regulation (hypothalamus), pancreatic beta cells that senseglucose and secrete insulin, and in the reproductive system.

Experiments in genetically altered mice that lack IRS2 or overexpressIRS2 reveal the essential role for IRS2 in peripheral insulin action andthe role of IRS2 in the function, growth and survival of pancreaticβ-cells. In mice, IRS2 is important for peripheral insulin action,pancreatic beta cell glucose sensing and insulin secretion, and CNScontrol of nutrient homeostasis that regulates appetite and obesity.Dysregulation of IRS2 signaling in peripheral insulin sensitive tissues,pancreatic beta cells and brain causes insulin resistance, β-cellfailure and obesity. Conversely, upregulation of IRS2 protein orincreasing its signaling potential or blocking pathways that inhibitsits function correct these problems, which prevents or cures the keypathologies of type 2 diabetes.

Drugs that increase IRS2 expression in mice stimulate beta cellfunction. An example of such a drug is exendin-4. Exendin-4 is a homologof the natural peptide produced in the intestine called glucagon-likepeptide-1. GLP1/Ex4 upregulates IRS2 levels in β-cells because theystimulate the production of cAMP. GLP1/Ex4 have similar effects in mouseand human islets to upregulate the levels of IRS2. Other drugs thatincrease cAMP levels in these and other cells also increase IRS2 levelsand stimulate its signaling pathways. Many of the positive actions ofGLPL1/Ex4 on mouse and human cells are prevented by blocking the IRS2gene from expressing its IRS2 protein product.

Thus upregulation of IRS2 function is a treatment for type 2 diabetesand other diseases in which dysregulated insulin signaling is acomponent, such as obesity, dyslipidemia, hypertension, cardiovasculardisease, Alzheimer disease, neurodegeneration, stroke, blindness, kidneydisease, female infertility, and angiogenesis disorders. Conversely,reduction of IRS2 function can be used to treat metastatic disorders(e.g., neoplastic diseases and cancer).

By upregulation of IRS2 function is meant an increase in the amount ofIRS2 protein within a cell or enhancing IRS2-mediated signaltransduction by activators as defined herein. By activator or inhibitorof IRS2 is meant a small molecule that binds to IRS2 alone and activatesor inhibits the signaling function of IRS2, or a small molecule thatbinds to a complex comprising IRS2 and other cellular proteins andwherein said small molecule cannot bind to the non-IRS2 proteins in theabsence of IRS2. By reduction or downregulation of IRS2 function ismeant a decrease in the amount of IRS2 protein within a cell or reducedIRS2-mediated signal transduction by inhibitors as defined herein.

There are three important elements of the invention disclosed hereinwhich may be described as follows:

1. The key concept pertains to modifying (i.e., stabilizing orinhibiting) the IRS2 binding interaction with various proteins bothupstream and downstream that interact with (bind to) IRS2. Theseinclude, for example, the human Insulin Receptor (HIR) which binds toand phosphorylates IRS2, the N-terminal c-jun kinase (JNK), PKCisoforms, ERK1 or ERK2, as well additional upstream or downstreamsignaling elements such as src homology 2 (SH2) domain-containingproteins that bind to IRS2 and may also phosphorylate, dephosphorylateor otherwise modify IRS2.

2. The specific pattern of covalent modifications of IRS2 such asphosphorylation of serine, threonine and tyrosine residues,ubiquitination patterns, or other covalent modifications that alter thefunction, intracellular localization, or stability of IRS2.

3. Methods that control the expression of the IRS2 gene in specificcells, including beta cells, brain cells, liver cells muscle cells,reproductive cells and tissues involved in reproduction, fat cells,mammary cells, bone cells and immune system cells, essentially any cellsof the body where IRS2 might be naturally or unnaturally expressed.

Accordingly, methods of restoring or enhancing insulin sensitivityand/or pancreatic β-cell function depend on increasing IRS2 function byany means, particularly those listed above. Thus, IRS2 function isincreased by increases in the amount of IRS2 protein in a cell, bymodulation of specific interactions between IRS2 and other cellularcomponents that modify IRS2, and by modulation of IRS2 interaction withits substrates.

Although certain of these effects may be opposite in nature dependingupon the cellular context, such modulations may be achievedpharmacologically with compounds, (and especially small molecules), thateither stabilize IRS2 interactions with other proteins or accelerate the“off” rate of such interactions after IRS2 has interacted with saidproteins. Depending upon the cellular context, any of the aforementionedactivities will lead to alterations in cellular functioning of theIRS2-mediated signal transduction cascades, resulting in improvements incellular signaling relevant to the disease states of interest as will bediscussed in detail below.

The method involves up regulating the expression or functional cellularactivity of IRS2, and preferably with respect to IRS1 or other IRSfamily members or other proteins. Up regulation of the IRS2 gene or IRS2protein function promotes cell and tissue functions particular to thespecific target tissue. Methods that promote IRS2 signaling, by upregulating IRS2 expression or IRS2 function in specific tissues cantarget or prevent specific diseases involving those specific tissues orcells. For example, up regulation of IRS2 in pancreatic β-cells improvesglucose stimulated insulin secretion. Drugs that up regulate the IRS2gene or promote IRS2 signaling in β-cells will promote β-cell functionand prevent or cure diabetes. Further, the level or functional activityof IRS2 can be modulated in human beings and other mammals in order toameliorate or even prevent the failure or massive destruction ofpancreatic β-cells that causes certain forms of diabetes, and reduce theneed for insulin by peripheral insulin sensitive tissues.

IRS2 is also important in peripheral tissues that respond to insulin, soup regulation of the IRS2 gene or up regulation of IRS2 signalingfunction makes tissues more sensitive to insulin and thus less insulinis needed to elicit the appropriate response. In one embodiment two ormore different drugs might be developed that promote IRS2 geneexpression or IRS2 function in β-cells or in hepatocytes or in neurons.Alternatively, a single compound might promote IRS2 gene expression orIRS2 signaling and function in all of these tissues. These effects ofIRS2 work together to keep glucose under control and prevent diabetesand related disorders that are modulated by IRS2 function.

In another example, up regulation of IRS2 expression or an increase ofIRS2 signaling function can also be beneficial to other tissues. Forexample, approximately half of the growth of a mouse brain depends onthe expression of the IRS2 gene. Therefore, drugs that promote IRS2signaling will also be expected to promote brain growth in mammals andpeople. IRS2 signaling also plays a role in dephosphorylation of the Tauprotein, a marker of Alzheimer disease. Up regulation of IRS2 in thehippocampus should promote normal function and contribute to theprevention of the neuronal degeneration associated with Alzheimerdisease.

IRS2 signaling also plays a role in feeding behavior and femalefertility. Mice lacking IRS2 tend to gain weight as a result of theinability of the brain to properly assess whether insulin has beensecreted or not after a meal, so the brain can not determine whether ameal has in fact been consumed. Upregulation of IRS2 in thehypothalamus, and particularly the arcuate nucleus of the hypothalmus,will promote appetite regulation that results in reduced weight gain oreven weight loss

The invention further comprises methods to discover and utilizecompounds that upregulate the function or levels of IRS2 in people toprevent or cure disesase associated with insulin resistance syndrome,especially diabetes.

In another embodiment, the invention can be used to determine whetherknown drugs already in use for the treatment of other diseases alsopromote IRS2 signaling functions or up regulation of IRS2 geneexpression. This would reveal new mechanisms of action for old drugsthat might indicate their use in human diseases caused by failure of theIRS2 signaling system, such as insulin resistance, diabetes and thecomplications resulting from these disorders.

IRS2 promotes growth of the retina. Mice lacking IRS2 display increasedloss of retinal neurons, especially rod and cones, leading to blindness.Thus, upregulation of IRS2 or increased IRS2 is useful for reducing orpreventing retinal degeneration and promoting retinal growth andregeneration.

Assay Systems for Identification and Subsequent Use of Modulators ofIRS2 Function.

Cell-based assay systems capable of being adapted specifically for theexamples which follow below have been previously developed by Applicants(See, for example, U.S. Pat. No. 5,688,655). Furthermore, certaincell-free assay systems are also useful for identifying compounds asdiscussed in detail in the examples given below. One such cell-freesystem consists of an electrochemiluminescence methodology wherebyprotein-protein interactions maybe measured by the emission of light ata specific wavelength when the IR interacts with (i.e. binds to) IRS2.Such cell free assay systems are also capable of being utilized in theidentification and characterization of compounds as discussed in detailin the examples given below. Other examples are well known toinvestigators of skill in the art.

Methods of Identifying and Using Compounds that Inhibit the Degradationof IRS2 in β-Cells.

In the most general approach, cell-based screens can be established toidentify compounds that block the intracellular degradation of IRS2.Although a variety of cell types can be used for this process, one thatexpresses (or overexpresses) IRS2 would be preferable, for example,according to prior teachings of U.S. Pat. No. 5,688,655 and relatedpatents. For example, ubiquitination promotes degradation of both IRS1and IRS2. Therefore, certain drugs that inhibit ubiquitination would beanticipated to protect cells from the deleterious effects that resultfrom the loss of IRS2 and can be identified using cell-based screens.Also, engineering an IRS2 cDNA for the purpose of detecting degradationof IRS2 would also be useful. This may be performed, for example,through the addition of a flag tag at the COOH end of the molecule. Suchtags include a FLAG tag, GFP tag, a MYC tag, and others known to thoseof skill in the art.

Methods of Identifying and Using Compounds that Upregulate IRS2 Functionin β-Cells.

Another way to upregulate IRS2 expression is by a substance thatstimulates transcription of the IRS2 gene. This can also be performedwith the cell based screening methods described above. β-cell lines,such as Min6, can be prepared with an IRS2 promoter linked to an easilydetectable reporter such as a green fluorescence protein (GFP) tofacilitate high throughput screening. PCR based screening methods mayalso be used to directly detect the expression of the endogenous gene.The hits can be tested for function on isolated mouse or human isletcells. Tissue specificity of the hits can be tested across various celllines to determine whether the identified compounds are specific forβ-cells or also promote IRS2 expression in other cells. GLP1 or exendin4can be used as positive controls in such an assay to validate the assay.

Upregulation of IRS2 in Beta Cells and Other Tissues Using Glucagon LikePeptide 1 (GLP-1).

GLP-1, as well as stable analogs like Exendin-4, has been suggested aspotential treatments for diabetes because they appear to promote insulinsecretion in response to increases in plasma glucose levels. GLP-1 hasbeen shown to reverse the age-dependent decline in β-cell function inrats or mice. Furthermore, GLP-1 also stimulates β-cell proliferationand neogenesis, and reduces or eliminates apoptosis of β-cells. GLP-1secretion has also been shown to decrease in people with Type 2diabetes, whereas subcutaneous administration of GLP-1 is able toimprove glucose homeostasis, lower body weight, reduce circulatingplasma free fatty acid and hemoglobin A(1C) and increase cellularresponsiveness to insulin. GLP-1 strongly up regulates IRS2 in betacells and other cells. Many of the positive effects of GLP-1 on betacells function are lost upon inhibition of IRS2 expression,demonstrating that IRS2 plays an important role in GLP-1 action (FIG.1). Interestingly, attempts to restore normal glucose homeostasis inIRS2 mice by the administration of Exendin-4 by injection wereunsuccessful.

Compounds and Methods of Using GLP-1 Analogs that Upregulate IRS2 inBeta Cells.

Since GLP-1 promotes expression of IRS2, small molecules (i.e. chemicalagents with molecular weights less than or equal to 1,000 atomic massunits (Daltons)) that are chemical analogues of GLP-1 or other chemicalagents capable of activating the GLP-1 receptor will also be able toincrease IRS2 expression. Such chemical compounds will preferably beorally available and can be used to promote IRS2 expression in β-cellsand other cells that contain GLP-1 receptors. By definition, substancescapable of upregulating IRS2 function are small molecules that increasethe level of IRS2 protein capable of functioning within a cell as wellas activators of IRS2 as defined herein.

Compounds and Methods of Using Cyclic AMP Modulators to Upregulate IRS2in Beta Cells and Other Tissues.

Drugs that upregulate the concentration of cAMP in cells are well known.Since IRS2 expression is modulated in part by cAMP levels in some cells,many current drugs may exert some of their effects by functioningthrough the upregulation of IRS2. Thus, evaluating known drugs forspecific effects on IRS2 expression in tissues will reveal new uses ofthe known drugs for the treatment of disease related to the loss of IRS2expression or cellular function. Some of the effects of certain knowndrugs may be dependent upon their ability to upregulate IRS2. Cell-basedassays based on IRS2 expression or reporter constructs can be used toevaluate known drugs or identify new compounds that upregulate IRS2expression or protein synthesis.

Methods of Identifying and Using Compounds that Upregulate IRS2 in aTissue-Specific Manner to Prevent Diseases of Insulin ResistanceSyndrome.

A drug that upregulates IRS2 might work on all tissues of the body, ordisplay tissue specificity. The effect(s) of known and unknown drugs ontissue-specific expression of IRS2 can be assessed. As known to one ofskill in this art, one method to do this is to construct a mouse thatexpresses an IRS2 construct containing a carboxy terminal (COOH)extension comprising green fluorescent protein. After administration ofcompounds to the test animal, all tissues can be evaluated forexpression of the tagged IRS2 protein to establish the tissue-specificeffects of the particular compound with respect to the expression of theIRS2 gene.

Methods of Identifying and Using Compounds that Stimulate Signaling byIRS2 in β-Cells and Other Cell Types.

IRS2 signaling is inhibited by many pathways including degradation andserine phosphorylation (FIG. 1). Compounds that inhibit these processeswill upregulate IRS2 function. As discussed previously, a general cellbased assay system can be set up to identify compounds that increaseIRS2 signaling. Various experimental strategies may be used to determinewhen such drugs are identified, including glucose uptake by the cell orthe subsequent expression of other known downstream genes. The effect ofthe compounds on the ability of insulin to modulate gene expression (forexample, in cells capable or incapable of expressing IRS1 or IRS2) canbe further determined. If a known set of genes are selected for thispurpose, reporter constructs can be developed and expressed in testcells. Alternatively, the entire genome can be used to assess theeffects of the compounds on these cells, in which case microarraytechnology can be employed.

Inhibitors of Serine Phosphorylation of IRS2.

Serine phosphorylation sites are known to modulate the activity of IRS1and IRS2 function. Compounds that inhibit or stimulate phosphorylationof the serine residues can be identified and these used to promote IRS2function and prevent diabetes, the complications of diabetes or theinsulin resistance syndrome, or other disorders in which IRS2 functionplays a role as discussed previously. A proteomic approach can bedeveloped to evaluate the phosphorylation of the relevant serineresidues. One approach could use antibodies that specifically recognizeand bind to phosphorylated serine residues in specific defined sequencemotifs. Specific antibodies can be generated in rabbits or in mice(polyclonal or monoclonal) and arrayed in such a way as to evaluate allthe residues simultaneously in cell extracts exposed to test compounds.

Introduction of an Artificial IRS2 Gene into β-Cells to Upregulate IRS2Expression.

Gene therapy is a general method for correcting errors in geneexpression in various cells. An artificial gene encoding IRS2 can beintroduced in β-cells to increase the expression of IRS2 and preventdiabetes. The gene can be constitutively active or it might containregulatory elements that control its expression. Viral vectors(adenovirus, HIV, lentivirus) and other delivery systems methods areavailable for transfection and upregulation of IRS2 in β-cells or othercells of the body. The upregulation of the IRS2 gene might beaccomplished during incubation of isolated human islets immediatelyafter isolation from human donors. These islets that are engineered toexpress IRS2 can then be used for transplantation. Upregulation of IRS2in murine islets promotes their function during transplantation,resulting in, for example, improved efficacy of transplants andreduction in the number of cells required.

Introduction of a Regulatory Sequence that Targets and Upregulates theEndogenous IRS2 Gene in β-Cells.

Gene expression is regulated by regulatory elements that bind varioustranscription factors. Regulatory elements may be substituted or addedto modify IRS2 gene expression. For example, regulatory elements thatincrease expression or that respond to other signaling systems can beused. Various strong promoters are available that can be inserted infront of IRS2 to increase expression. Such elements can be targeted tocells in a cell type- or tissue-specific manner.

Introduction of an Artificial IRS2 Gene into Isolated β-Cells orPluripotent Stem Cells for Implantation into Patients.

Islet cells can be removed from the body and treated with DNA that willalter the expression of the IRS2 gene, such as by introducing additionalcopies of IRS2, or by altering the regulatory region of IRS2 byhomologous recombination. These engineered cells can then be replacedinto the body to cure diabetes. Alternatively, islet cells can beobtained from other sources (e.g., matched donors), expression of IRS2upregulated by altering the expression of the gene by the methodsdescribed above and the cells can be replaced in the patient. In anotherapproach, islet cells can be differentiated from isolated stem cells.Islets cells can also be obtained from other mammals (e.g., pigs, cows,primates) and expression of IRS2 increased by genetic means as describedabove. The islet cells can be modified so as to be acceptable for humantransplantation, or placed into an appropriate biocompatible containerto avoid rejection.

Introduction of an Artificial IRS2 Gene into Neurons that areSubsequently Put Back into Patients.

IRS2 is highly expressed in specific regions of the brain, including thehypothalmus, hippocampus, amygdala and cortex. IRS2 promotes neuronalgrowth and inhibits the phosphorylation of Tau, a marker of Alzheimer'sdisease. IRS2 also promotes growth of CNS neurons during development.Upregulation of IRS2 expression in cells used to repair neuronal damagecan significantly enhance the opportunity for repair and the productionof neurotransmitters. Introduction hippocampal neurons expressing IRS2into a hippocampus might prevent Alzheimer disease in susceptibleindividuals. Neurological damage in general, caused by trauma, might berepairable by introducing neurons into the damaged area for repair. Therepair might be more likely to succeed if the neurons used containelevated levels of IRS2 expression. Elevated IRS2 expression can beachieved by genetic manipulation or pharmaceutically active agents asoutlined above.

Methods of Identifying and Using Compounds that Inhibit Degradation ofIRS2 in β-Cells.

IRS2 is sensitive to proteolytic degradation. Accordingly, proteaseinhibitors can be used to upregulate IRS2 signaling potential byinterfereing with degradation of IRS2. Cell based or cell-free screeningassay systems (as previously described) screens can be used to identifysuch inhibitors.

Methods of Identifying and Using Compounds that Block the Interaction ofIRS2 with Degrading Enzymes in β-Cells and Other Cell Types.

Specificity is frequently achieved in biological systems throughspecific protein-protein interactions. In the case of enzymes thatpromote degradation of IRS2, compounds that prevent the specificinteraction between IRS2 and the degradation enzymes would result in theupregulation of IRS2 protein. Cell-based and cell-free screens can bedesigned using tagged proteins to identify proteins that prevent theinteraction of IRS2 with degradation enzymes.

Enhancement of Fertility.

Female mice lacking IRS2 are infertile. By upregulating IRS2 signalingor IRS2 gene expression in ovaries, ovulation may be enhanced.

Methods of Identifying and Using IRS2 Promoting Compounds to ReverseCatabolism During Acute Trauma.

Insulin resistance is a major problem during acute trauma. Decreasedinsulin secretion during acute trauma exacerbates the problems. Insulinresistance and decreased insulin secretion leads to massive catabolismthat can threaten survival in the early period of repair. Both processescan be explained by the loss of IRS2 signaling due to inhibition byinflammatory processes. Drugs that promote IRS2 function, prevent IRS2degradation, or promote IRS2 expression will reverse these effects.

Methods of Identifying and Using IRS2 Promoting Compounds to PreventInsulin Resistance and Diabetes Associated with Obesity.

A major problem with obesity is that peripheral tissues become insulinresistant; if β-cells fail to make enough insulin to overcome theinsulin resistance then diabetes develops. This can be treated withcompounds that upregulate IRS2 in β-cells and/or peripheral tissues.Upregulating IRS2 in β-cells promotes better glucose detection andinsulin secretion, and upregulating IRS2 in peripheral tissues reducesthe insulin requirements. Accordingly, the incidence of life threateningcomplications of obesity can be reduced.

Regulation of Genes that Modulate IRS2 Levels and Function in β-Cellsand Other Cell Types.

Like other genes, IRS2 is regulated by transcription factors such asCREB. One way to increase IRS2 expression is to increase the activity ofthe transcription factors that stimulate the transcription of the IRS2genes. Such compounds can be easily identified through the use of cellbased screens, as previously described. In particular, cells can beengineered to express IRS2 reporter genes and high or low levels ofCREB. Substances that up regulate the IRS2 reporter can then beidentified by measuring the differential responses of the compounds incells expressing high or low levels of CREB.

Identification of Compounds that Activate or Inhibit Components of theIRS2 Signaling Cascade.

IRS2 is an early component in a signaling cascade that controls cellularevents and gene expression (FIG. 1). Many other components of thepathway are known. Accordingly, reduced activity of IRS2 may becompensated by modulating activity of downstream components. Forexample, genetic deletions of pTEN, Foxo1 or PTP1B, all of which inhibitIRS2 signaling, compensate for the lack of IRS2 and promote β-cellfunction in mice; Accordingly, substances that inhibit these componentscan stimulate IRS2 signaling pathways. Such drugs can be identified bycell based or purified protein-based assays. Similar strategies can beapplied to other elements in the cascade (FIGS. 1 and 2).

Methods of Identifying and Using Compounds that Suppress Inhibition orDestruction of IRS2 in β-Cells by the Immune System.

Type 1 diabetes is an autoimmune disease. Leukocytes are attracted toislets by β-cell autoantigens. Once they have migrated to pancreaticislets, the leukocytes then attack and destroy β-cells through cell-cellcontacts or by releasing proinflammatory cytokines that promote β-celldeath. Death of a β-cells is thought to occur through mechanisms thatare common to other cells, such as activation of the caspase cascadeincluding the cleavage and activation of caspase-3. IRS2 signalinggenerally inhibits apoptosis of many cells types, including β-cells, bypromoting phosphorylation of BAD and dissociation of BCL1 that inhibitsa cascade that culminates in caspase-3 cleavage and activation. One ofthe ways that leukocytes prepare cells for rapid killing by promotingdegradation of IRS2 or inhibiting its function. Compounds that inhibitdegradation of IRS2 and inhibit its serine phosphorylation will opposethe killing effects of leukocytes. These compounds can be identified byestablishing cell based assays performed in the presence ofproinflammatory cytokines. Compounds that upregulate IRS2, stabilize it,promote its function or block its interaction with proteins in theinflammatory cascades will promote β-cell survival and function that canbe assayed with standard tools.

Methods and Compounds that Protect the Tyrosine Phosphorylation State ofIRS2 in β-Cells.

IRS2 mediates signals that promote functional growth and survival ofβ-cells through tyrosine phosphorylation mediated by the IGF1 receptor,insulin receptor or other receptors coupled to tyrosine kinases.Phosphatases dephosphorylate IRS proteins and inhibit these positiveeffects. Compounds that inhibit specific phosphatase activities inβ-cells will upregulate IRS2 function and promote β-cell function.General screening methods are well known that can be used to find drugswith the proper phosphatase inhibiting effects. For example PTP1B is anexample of one such phosphatase that can be targeted for inhibition, andthere are other important phoshatases in β-cells as well as in othercell types.

Methods and Compounds that Inhibit Ubiquitination of IRS2 in β-Cells.

IRS2 proteins are targeted for degradation upon ubiquitination.Therefore, compounds that inhibit the interaction between IRS2 andubiquitin transferase complexes or inhibit the accessibility of residuesthat get ubiquitinated will prolong the half life of IRS2 and therebyenhance is signaling capacity.

Methods and Compounds that Activate or Inhibit Serine, Threonine andTyrosine Phosphorylation of IRS2 in β-Cells, Neurons, and Other CellTypes that are IRS2 Sensitive for Growth, Function or Survival.

IRS2 proteins are targets for phosphorylation by serine, threonine andtyrosine kinases. Therefore, compounds that inhibit or stimulatephosphorylation of IRS2 will modulate IRS2 cellular function in atherapeutically useful manner. Such functions of IRS2 will include itsability to interact with (bind to) other proteins involved in varioussignal transduction cascades that are beneficial for the treatment ofhuman diseases such as type 2 diabetes, neurodegenerative diseases suchas Alzheimer's disease, cardiovascular diseases, peripheral neuropathy,vascular disease, retinopathies, macular degeneration, and the like.

In the present invention, a therapeutically effective amount of one ormore compounds that modulate IRS functional activity is administered toa mammal in need thereof. The term “administering” as used herein meansdelivering the compounds of the present invention to a mammal by anymethod that may achieve the result sought. They may be administered, forexample, orally, parenterally (intravenously or intramuscularly),topically, transdermally or by inhalation. The term “mammal” as usedherein is intended to include, but is not limited to, humans, laboratoryanimals, domestic pets and farm animals. “Therapeutically effectiveamount” means an amount of a compound that, when administered to amammal, is effective in producing the desired therapeutic effect, suchas inhibiting kinase activity.

Suitable carriers include, for example, one or more of water, saline,phosphate buffered saline, dextrose, glycerol, ethanol and the like, aswell as combinations thereof. Carriers can further comprise minoramounts of auxiliary substances, such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the binding polypeptides. The compositions can, as is well known inthe art, be formulated so as to provide quick, sustained or delayedrelease of the active ingredient after administration to the mammal.

The compositions of this invention can be in a variety of forms. These,include, for example, solid, semi-solid and liquid dosage forms, such astablets, pills, powders, liquid solutions, dispersions or suspensions,liposomes, suppositories, injectable and infusible solutions. Thepreferred form depends on the intended mode of administration andtherapeutic application.

Such compositions of the present invention are prepared in a manner wellknown in the pharmaceutical art. In making the composition the activeingredient will usually be mixed with a carrier, or diluted by a carrierand/or enclosed within a carrier which can, for example, be in the formof a capsule, sachet, paper or other container. When the carrier servesas a diluent, it can be a solid, semi-solid, or liquid material, whichacts as a vehicle, excipient or medium for the active ingredient. Thus,the composition can be in the form of tablets, lozenges, sachets,cachets, elixirs, suspensions, aerosols (as a solid or in a liquidmedium), ointments containing, for example, up to 10% by weight of theactive compound, soft and hard gelatin capsules, suppositories,injection solutions, suspensions, sterile packaged powders and as atopical patch.

It should be appreciated that the methods and compositions of thepresent invention can be administered to any suitable mammal, such as arabbit, rat, or mouse. More preferably, the mammal is a human.

Throughout this application, various publications, patents, and patentapplications have been referred to. The teachings and disclosures ofthese publications, patents, and patent applications in their entiretiesare hereby incorporated by reference into this application to more fullydescribe the state of the art to which the present invention pertains.

It is to be understood and expected that variations in the principles ofinvention herein disclosed may be made by one skilled in the art and itis intended that such modifications are to be included within the scopeof the present invention.

The examples which follow further illustrate the invention, but shouldnot be construed to limit the scope in any way.

Examples

The knockout of IRS2 shows that insulin action and secretion are closelylinked at the molecular level through the insulin receptor substrate-2(IRS2) branch of the insulin/IGF signaling cascade. Mice lacking IRS2display similarities to humans with type 2 diabetes. The actions ofβ-cell-specific overproduction of IRS2 on diabetes development andpancreatic β-cell function in murine models of autoimmune diabetes andislet transplantation were evaluated. (Hennige, A. et al., J. Clin.Invest. 112:1521-1531 (2003)). M. White and colleagues showed thatupregulation of IRS2 in pancreatic β-cells promotes β-cell growth,survival, and insulin secretion. Furthermore, they demonstrated thatincreased expression of IRS2 in β-cells improves islet transplantation,as significantly fewer islets were required to normalize serum glucoselevels. The data support a pharmacological role for IRS2 or downstreamfactors in the treatment of β-cell failure and human diabetes. Theresults provide support for the teachings of the present invention thatone way to realize the goal of preventing or curing diabetes and relatedmetabolic disorders is to identify compounds that increase the activityor function of IRS2 in β-cells, or in other tissues of the body thatrequire the IRS2 signaling function including brain or specific neuronaltissues like retina, hypothalamus, hippocampus, amygdala; heart, liver,muscle, adipose, ovary, pituitary, leukocytes, retina, or other cells ortissues that respond to insulin or IGF1 by generating signaling cascadesthat are coordinated by IRS2.

Jhala et al. (Genes Dev. 17:1575-1580 (2003)) provides evidence thatIRS2 is a CREB responsive gene, and that GLP1/ex4 strongly increasesIRS2 expression in β-cell lines, presumably through cAMP mediatedphosphorylation of CREB. By contrast, IRS1 expression is not regulatedthrough activation of CREB. The results demonstrate that compounds existthat can upregulate the synthesis of IRS2 in cells, and particularlywith respect to another IRS protein, and that compounds that increasecAMP levels in cells, including cell surface receptor agonists,phosphodiesterase inhibitors, PKA activators, and cAMP mimetics, canalso upregulate IRS2 through activation of Creb.

Accordingly, these results taken together with the teachings of theinvention described herein provide additional evidence that IRS2synthesis can be increased in a specific manner in β-cells to prevent orcure diabetes.

1.-15. (canceled)
 16. A method of determining whether a small moleculeis an activator or an inhibitor of IRS2 which comprises: a) providing aTest Cell which overproduces IRS2 and exhibits an increase in binding ofan IRS2-binding protein to IRS2, relative to a Control cell whichproduces IRS2 at a lower level, or does not produce the protein at all,and which exhibits a lesser amount of binding of said protein to IRS2;b) causing the small molecule to come into contact with the intact TestCell; c) measuring the amount of the IRS2 binding protein bound to IRS2.17. A method of identifying a small molecule capable of increasing thelevel of expression from an IRS2 promoter in a mammalian cell whichcomprises: a) providing a Test Cell which contains said IRS2 promoteroperably linked to a reporter gene such that increased expression of theIRS2 promoter sequence using a substance known to be capable ofupregulating the endogenous IRS2 gene results in an increase in reporterprotein levels; b) causing said small molecule to come into contact withthe intact Test Cell, and c) determining whether an increase in reporterprotein level in the Test Cell has occurred.